The technique of DNA fingerprinting is recognised to be capable of "revolutionising forensic biology".
The power of DNA fingerprinting technology is its potential to positively discriminate between closely related individuals and to determine pedigree. The probability that two related human individuals share the same DNA fingerprint is 1 in 100 million, except if they are identical twins. For unrelated individuals, the odds are even higher.
Apart from humans, horses are the other major species for which conventional blood-typing is widely established and accepted. Countries maintaining a stub book register of thoroughbred horses use about 20 polymorphic blood-typing systems to establish the identity of individuals and their pedigree. However, up to 4% of parentage cases of horses can not be satisfactorily resolved by these conventional blood-typing techniques.
Genetic fingerprinting consists broadly of the following aspects:
(a) Basis of genetic fingerprinting
It has been known for some years that there are hypervariable regions of DNA which show multiallelic variation. Those variable regions consist of tandem repeats of a short sequence, termed "minisatellites" which are stably inherited. Differences in the number of repeats provide the basis of polymorphism. Dr. Alec J. Jeffreys had discovered that many of such minisatellites have a high degree of homology and that certain core sequences are capable of seeking out of hybridising to more than one minisatellite. Such core sequences provide a powerful probe for revealing polymorphism and provide unique "fingerprints" for each individual.
(b) Application of the probe
Utilisation of probes involves the preparation of DNA in a suitable form and medium. The extracted DNA is cut by a suitable restriction enzyme into fragments of varying sizes. These fragments are separated by size by gel electrophoresis. The DNA fragments are then denatured and may or may not be transferred to a suitable medium by "blotting". The probe which has been tagged with a suitable marker or label is applied to the denatured fragments. The marker will then show up regions of hybridisation as bands which form the "fingerprint" of the DNA.
(c) Preferred criteria of a good DNA fingerprint
(i) Bands should preferably be clearly visible.
(ii) Bands should preferably be distinct and clearly defined (from each other)
(iii) All bands should preferably be traceable to each parent.
(iv) In pedigree testing, there should preferably be at least 6 bands from each parent.
(v) The pattern of bands should preferably be specific or unique to each individual (except where the individual is an identical twin).
(vi) The bands should preferably be produced in DNA fragments greater than 1000 bp in length.
(vii) The bands should preferably be capable of detection using probes carrying both radioactive and non-radioactive labels.
(viii) The bands should preferably be capable of detection with less than saturating concentrations of probe.
(ix) The bands should preferably be capable of being produced from picogram quantities of DNA using DNA amplification procedures.
(d) Existing families of probes
It is suggested that the human genome might contain at least 1500 hypervariable regions (Jeffreys, A. J. (1987), Biochemical Society Transactions 15: 309-317). In UK Patent specification GB 2 166 445A, there is disclosed a family of probes derived from the 33 bp sequence in an intron of the human myoglobin. Other families of probes have been reported:
(i) a bacterium, Escherichia coli `chi` sequence (Jeffreys, et, al, (1985) Nature 314: 67-73.
(ii) a bacteriophage, M13 sequence (Vassart, G., Georges, M., Monsieur, R., Brocas, H., Lequarre, A. S. & Christophe, D. (1987). Science, 235: 683-684).
(iii) a "simple quadruplet repeat (sqr)" sequence from snakes and other animals (Ali. S., Muller, C. R. and Epplen, J. T. (1986) Human Genetics 74: 239-243).
(iv) a human DNA library (Wyman, A. & White, R., (1980) Proc. Nat'l. Acad. Sci., U.S.A., 77: 6754-6758; Nakamura Y., Leppert, M., O'Connell, P., Wolff, R., Holm, T., Culver, M., Martin, C., Fujimoto, E., Hoff, M., Kumlin, E. and White, R. (1987) Science, 235: 1616-1622).
(v) a DNA sequence close to the human insulin gene (Bell G. I., Selby, M. J. and Rutter, W. J. (1982) Nature 295: 31-35).
(vi) DNA sequences close to the alpha-related globin genes, including myoglobin and zeta-globin (Proudfood N. J., Gil, A. & Maniatis, T. (1982). Cell, 31: 223-563; Goodbourne S. E. Y., Higgs, D. R., Clegg, J. B. and Weatherall, D. J. (1983) Proc. Natl. Acad, Sci., U.S.A., 80: 5022-5026; Higgs, D. R., Goodbourn, S. E. Y., Wainscoat, J. S., Clegg, J. B. and Weatherall, D. J. (1981). Nucleic Acids Res. 9: 4213-4224).
(vii) Sequences near the human Harvey-ras oncogene (Capon D. J., Chen E. Y., Levinson, A. D., Seeberg, P. H. and Goeddel, D. V. Nature, 302: 33-37).
(viii) the X-gene region of the hepatitis B virus (Nakamura Y., Leppert, M., O'Connell, P., Wolff, R., Holm, T., Culver, M., Martin, C., Fujimoto, E., Hoff, M., Kumlin, E. and White, R. (1987) Science, 235: 1616-1622).
(ix) Drosophila `Per` gene (Shin, H. S., Bargiello, T. A., Clarke, B. T., Jackson, F. R. and Young, M. W. (1985) Nature 317: 445-4419; Georges, M., Lequarre, A-S, Castelli, M., Hanset, R. & Vassart, G. (1988) Cytogenetics & Cell Genetics, 47: 127-131).
(x) human satellite III sequence (Fowler, C., Drinkwater, R., Burgoyne, L., Skinner, J. (1987) Nucl. Acids Res. 15: 3929).
Some of these probes have been tested in relation to humans with different results and degrees of success under similar conditions (Vassart, 1987, supra). The resulting DNA fingerprint is sensitive to the sequence of the repeats and within a family, different probes which vary slightly in their `core` sequence detect almost completely different sets of hypervariable regions to produce different fingerprint patterns (Jeffreys 1987, supra).
(e) Cross-species hybridisation
It is recognised that the genetic fingerprinting technique is not limited to human use. Cross-species nucleic acid hybridisation is well-known and various families of probes196ve been used on species other than that from which the probe was derived. In the UK Patent Specification 2, 166, 445A, it was stated that the probe derived from the human myoglobin gene has been tested in economically-important animals such as dogs, cats, sheep, pigs, horses and cattle with varying degrees of success. In particular, it was stated that the fingerprint obtained from the use of the most promising probe on horses and pigs "are faint [and] contain very few bands compared with the corresponding human DNA fingerprints". This was also stated in Jeffreys, A. J., Hillel, J., Hartley, N., Bulfield D. B., Wilson, V. & Harris, S. (1987) Animal Genetics, 18 (Supplement 1): 141-142.